Oral delivery of insulin associated to polymeric nanoparticles in diabetic rats
Introduction
Since the parenteral administration is the only route of insulin delivery, alternative routes of administration (oral, nasal, rectal, pulmonary and ocular) have been extensively investigated [1]. Among them, the oral route seems to be the most convenient and physiological because insulin undergoes a first hepatic bypass, thus warranting a primary effect by inhibiting hepatic glucose output. However, insulin is strongly degraded by proteolytic enzymes in the gastrointestinal tract. In addition, this peptide is very poorly absorbed after oral administration. In order to protect it from biodegradation and to improve its intestinal absorption, insulin has been associated to antiproteases [2], hydrogels [3], or combined with absorption enhancers such as cyclodextrins [4], bile salts [5] and surfactants [6]. Insulin was also encapsulated in polymeric biodegradable nanocapsules, nanospheres or microparticles associated or not to sufactants or antiproteases [7], [8], [9]. Thiolated or mucoadhesive polymers of polyacrylic nature [10], [11], chitosan-coated liposomes [12] or liposomes encapsulated in alginate-chitosan gel capsules [13] also increased the residence time of insulin in the vicinity of intestinal absorptive cells allowing its absorption. The modification of the permeability of the intestinal mucosa to peptides by using zonula occludens toxin [14] or chitosan [15] allowed the paracellular uptake of insulin by opening the tight junctions between epithelial cells. The reduction of the mucous/glycocalyx layers over the intestinal epithelial cells by a treatment with hyaluronidase [16] also increased the absorption of insulin. It has also been suggested to chemically modify the insulin molecule [17], [18] in order to increase its solubility and to improve its stability against gastrointestinal enzymes. From these different studies, it can be concluded that the oral absorption of insulin has been demonstrated. Nevertheless the possible toxicity of all the excipients has not yet been proved.
Therefore, we developed insulin nanoparticles prepared with well accepted polymers: a biodegradable polymer, poly(ε-caprolactone) used for the manufacturing of resorbable threads and, second, a polyacrylic polymer, Eudragit® RS, widely used for the formulation of conventional solid dosage forms (i.e. coating of tablets). In addition, owing to its polycationic nature, this latter polymer could favor mucoadhesion such as chitosan [19]. In this study, nanoparticles were manufactured according to the double emulsion technique mainly used for peptides and proteins. After physico-chemical characterization (diameter, zeta-potential, insulin loading), the biological efficacy of insulin-loaded nanoparticles was determined after oral administration in diabetic rats. In addition and in order to potentially elucidate the absorption mechanisms, the intestinal uptake of fluorescent labeled insulin encapsulated into these nanoparticles was studied by fluorescence microscopy.
Section snippets
Material
Regular human insulin (Actrapid®) was kindly provided by Novo-Nordisk (Paris, France). Poly(-ε-caprolactone) (MW =42,000) was purchased from Sigma-Aldrich (L'Isle d'Abeau Chesnes, France) and Eudragit® RS (methacrylic acid esters with a small proportion of trimethylamonioethyl methacrylate chloride) (MW = 150,000) was a gift from Röhm Pharma (Darmstadt, Germany). Fluorescein isothiocyanate and polyvinylalcohol (PVA, MW = 30,000, 88% hydrolyzed) were obtained from Sigma-Aldrich. All other chemicals
Preparation of nanoparticles
The preparation of nanoparticles was carried out by the multiple emulsion technique previously described by Hoffart et al. [20]. Briefly, 1 mL of an aqueous solution of insulin (1000 IU) was first emulsified, by sonification for 30 s, in methylene chloride (10 mL) containing 250 mg of polymers (PCL/Eudragit RS 50/50). The resulting water-in-oil emulsion was thereafter poured into 40 mL of a polyvinyl alcohol aqueous solution (0.1%) and sonicated for 1 min, involving the formation of the second
Structure and characterization of nanoparticles
As shown in Table 1, unloaded and insulin-loaded nanoparticles showed a homogeneous size distribution with a mean diameter of 331 ± 11 nm and 358 ± 12 nm respectively, an overall positive charge and a high encapsulation efficiency (96.3 ± 0.42%).
Effect of subcutaneous administration of insulin nanoparticles on glycemia
As shown in Fig. 1, encapsulation of insulin prolonged the hypoglycemic effect of the drug. Indeed, after the s.c. injection of non encapsulated insulin (10 IU/kg), glycemia decreased significantly by 22% (p < 0.001) after 30 min, the maximal decrease (70%, p <
Discussion
This study clearly indicates that encapsulation of insulin into nanoparticles (prepared with a blend of a biodegradable polymer, poly(ε-caprolactone) and a polycationic non biodegradable polymer of a polyacrylic nature, Eudragit® RS), allows the preservation of its biological activity leading to i) its prolongation of action and ii) its oral absorption in a diabetic rat model. The former statement was demonstrated after subcutaneous administration of insulin-loaded nanoparticles in diabetic
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